Bacterial resistance to antibiotics has been an evolving problem since the dawn of the antibiotics era, requiring consistent scientific advances over the years in antibiotic discovery, epidemiological surveillance and infection control techniques to overcome the then-current clinical problem. One way to address resistance mechanisms is to attack the bacteria on many different fronts as has been done in the anti-viral field with HIV. However, unlike in the antiviral field, most commercial antibiotics were discovered many years ago and little advancement has been made in the discovery of novel therapeutics. This is even after the dawn of the genomics era when complete bacterial genomes were sequenced and unique enzymatic pathways identified. A multitude of targets have been screened by high-throughput screening methods to no avail. Recently, the reason for this high failure rate has been analyzed and conclusions drawn that traditional HTS libraries, designed to fit all disease indications, do not possess the properties required for anti-bacterial agents. Retrospective analysis reveal that in general successful antibacterial agents are more polar and larger than other drug molecules, and in fact, do not fit the criteria used to build most large HTS collections. Rather than re-building HTS libraries for antibacterial research which would be a tremendous and costly undertaking, in this proposal, we will use another method, fragment-based lead discovery, where fragments of drugs are screened rather than intact molecules. Because the compounds are smaller, the libraries need not be large or costly to assemble. Furthermore, as we find compounds that bind to our target and begin to increase the size of the fragments, we can design in antibiotic-friendly chemical properties at the same time we are building in potency. We are focusing on the bacterial cell wall synthesis pathway in gram negative bacteria, targeting two proteins: LpxA and LpxD. Both proteins are essential and because the cell wall can invoke resistance to some antibiotics, inhibitors may not only be therapeutic agents as a mono-therapy but could be co-dosed with existing resistant antibiotics. PUBLIC HEALTH RELEVANCE: Bacterial resistance to antibiotics has been an evolving problem since the dawn of the antibiotics era, requiring consistent scientific advances over the years in antibiotic discovery, epidemiological surveillance and infection control techniques to overcome the then-current clinical problem. We are addressing resistance mechanisms by finding inhibitors of bacterial cell wall synthesis in gram negative bacteria. We are targeting two proteins in the pathway: LpxA and LpxD using the method of fragment- based lead discovery. Traditional modern drug discovery methods have been largely unsuccessful in identifying antibacterial compounds primarily because we have been looking in the wrong place (in chemical space). Chemical properties of successful antibiotics have been identified and will be adhered to through the course of this study.